Porous honeycomb film membranes enhance endothelial barrier integrity in human vascular wall bilayer model compared to standard track-etched membranes.

In vitro vascular wall bilayer models for drug testing and disease modeling must emulate the physical and biological properties of healthy vascular tissue and its endothelial barrier function. Both endothelial cell (EC)-vascular smooth muscle cell (SMC) interaction across the internal elastic lamina (IEL) and blood vessel stiffness impact endothelial barrier integrity. Polymeric porous track-etched membranes (TEM) typically represent the IEL in laboratory vascular bilayer models. However, TEM stiffness exceeds that of diseased blood vessels, and the membrane pore architecture limits EC-SMC interaction. The mechanical properties of compliant honeycomb film (HCF) membranes better simulate the Young's modulus of healthy blood vessels, and HCFs are thinner (4 vs. 10 µm) and more porous (57 vs. 6.5%) than TEMs. We compared endothelial barrier integrity in vascular wall bilayer models with human ECs and SMCs statically cultured on opposite sides of HCFs and TEMs (5 µm pores) for up to 12 days. Highly segregated localization of tight junction (ZO-1) and adherens junction (VE-cadherin) proteins and quiescent F-actin cytoskeletons demonstrated superior and earlier maturation of interendothelial junctions. Quantifying barrier integrity based on transendothelial electrical resistance (TEER), membranes showed only minor but significant TEER differences despite enhanced junctional protein localization on HCF. Elongated ECs on HCF likely experienced greater paracellular diffusion than blocky ECs on TEM. Also, larger populations of plaques of connexin 43 subunit-containing gap junctions suggested enhanced EC-SMC communication across the more porous, thinner HCF. Compared with standard TEMs, engineered vascular wall bilayers cultured on HCFs better replicate physiologic endothelial barrier integrity.

Human Vascular Wall Microfluidic Model for Preclinical Evaluation of Drug-Induced Vascular Injury.

Drug-induced vascular injury (DIVI) in preclinical animal models often leads to candidate compound termination during drug development. DIVI has not been documented in human clinical trials with drugs that cause DIVI in preclinical animals. A robust human preclinical assay for DIVI is needed as an early vascular injury screen. A human vascular wall microfluidic tissue chip was developed with a human umbilical vein endothelial cell (HUVEC)-umbilical artery smooth muscle cell (vascular smooth muscle cell, VSMC) bilayer matured under physiological shear stress. Optimized temporal flow profiles produced HUVEC-VSMC bilayers with quiescent endothelial cell (EC) monolayers, EC tight junctions, and contractile VSMC morphology. Dose-response testing (3-30 µM concentration) was conducted with minoxidil and tadalafil vasodilators. Both drugs have demonstrated preclinical DIVI but lack clinical evidence. The permeability of severely damaged engineered bilayers (30 µM tadalafil) was 4.1 times that of the untreated controls. Immunohistochemical protein assays revealed contrasting perspectives on tadalafil and minoxidil-induced damage. Tadalafil impacted the endothelial monolayer with minor injury to the contractile VSMCs, whereas minoxidil demonstrated minor EC barrier injury but damaged VSMCs and activated ECs in a dose-response manner. This proof-of-concept human vascular wall bilayer model of DIVI is a critical step toward developing a preclinical human screening assay for drug development. Impact statement More than 90% of drug candidates fail during clinical trials due to human efficacy and toxicity concerns. Preclinical studies rely heavily on animal models, although animal toxicity and drug metabolism responses often differ from humans. During the drug development process, perfused in vitro human tissue chips could model the clinical drug response and potential toxicity of candidate compounds. Our long-term objective is to develop a human vascular wall tissue chip to screen for drug-induced vascular injury. Its application could ultimately reduce drug development delays and costs, and improve patient safety.

Drug Transcellular Transport Assay Using a High Porosity Honeycomb Film.

In vitro transport studies across cells grown on culture inserts are widely used for evaluating pharmacokinetic characteristics such as intestinal membrane permeability. However, measurements of the apparent permeability coefficient of highly lipophilic compounds are often limited by transport across the membrane filters, not by transport across the cultured cells. To overcome this concern, we have investigated the utility of a high-porosity membrane honeycomb film (HCF) for transcellular transport studies. Using the HCF inserts, the apparent permeability coefficient (Papp) of the drugs tested in LLC-PK1 and Caco-2 cells tended to increase with an increase in lipophilicity, reaching a maximum Papp value at Log D higher than 2. In contrast, using the commercially available Track-Etched membrane (TEM) inserts, a maximum value was observed at Log D higher than 1. The basolateral to apical transport permeability Papp(BL→AP) of rhodamine 123 across LLC-PK1 cells that express P-glycoprotein (P-gp) cultured on HCF inserts and TEM inserts was 2.33 and 2.39 times higher than the reverse directional Papp(AP→BL) permeability, respectively. The efflux ratio (Papp(B-A)/Papp(A-B)) of rhodamine 123 in LLC-PK1 expressing P-gp cells using HCF inserts was comparable to that obtained using TEM inserts, whereas the transported amount in both directions was significantly higher when using the HCF inserts. Accordingly, due to the higher permeability and high porosity of HCF membranes, it is expected that transcellular transport of high lipophilic as well as hydrophilic compounds and substrate recognition of transporters can be evaluated more accurately by using HCF inserts.

Excitatory actions of mushroom poison (acromelic acid) on unmyelinated muscular afferents in the rat.

Ingestion of a poisonous mushroom, Clitocybe acromelalga, results in strong and long-lasting allodynia, burning pain, redness and swelling in the periphery of the body. Acromelic acid (ACRO), a kainate analogue isolated from the mushroom, is assumed to be involved in the poisoning. ACRO has two isomers, ACRO-A and ACRO-B. The potency of ACRO-A is a million times higher than that of ACRO-B for induction of allodynia when intrathecally administered in mice. The effect of ACRO on the primary afferents of somatic tissues remains largely unknown. The aim of the present study was to examine the effect of ACRO-A on the response behavior of unmyelinated afferents in the skeletal muscle. For this purpose single fiber recordings of C-afferents were made from rat extensor digitorum longus (EDL) muscle-common peroneal nerve preparations in vitro. Intramuscular injections of ACRO-A at three different concentrations (10(-12), 10(-10) and 10(-8)M, 5 microl over 5s) near the receptive field in the EDL muscle elicited excitation of C-afferents (12%, 50% and 44%, respectively). ACRO-A at the concentration of 10(-10)M induced the strongest excitation. The incidence of ACRO-A responsive fibers at the concentration of 10(-10) and 10(-8)M was significantly higher than that at 10(-12)M. The responses to mechanical and heat stimulations did not differ between ACRO-A sensitive and insensitive fibers. These results clearly demonstrated the powerful excitatory action of ACRO-A on mechanosensitive unmyelinated afferents in the rat skeletal muscle.